Objectives To develop reagents for the improved diagnostic serology of Lyme disease. Results A segment (P7-1) of the variable domain of VlsE, the variant surface antigen of the spirochete Borrelia burgdorferi sensu lato was cloned and sequenced. and a conserved, immunodominant region of 26 amino acids (IR6) was identified. A 26-mer peptide (C6) was synthesized on the basis of the IR6 sequence and a peptide ELISA was developed for serodiagnosis of Lyme disease. Ten rhesus monkeys were infected with Borrelia burgdorferi strains JD1 or B31 by either tick or needle inoculation. Blood samples were periodically collected for up to 3 years post-infection (PI) until the animals were sacrificed, and serum samples were assessed for antibody responses to C6. Anti-C6 antibody was detectable in 6 monkeys as early as week 2 and in the remaining animals between weeks 3 and 5 PI. Antibody persisted at high levels for as long as serum samples were monitored. Forty-one serum samples from patients the majority of which had culture-confirmed, acute Lyme borreliosis, were obtained from the Centers for Disease Control and Prevention (CDC) and assessed with the C6 ELISA. Sensitivity was 85% (35/41), whereas with a commercially available Lyme disease ELISA it was 78% (32/41), and 49% (20/41) with a commercially available IgG Lyme immunoblot, 51% (21/41) with an IgM immunoblot, and 78% (32/41) by the combination of both IgG and IgM immunoblots. Of a group of 99 randomly collected serum samples from hospital patients in Louisiana, where Lyme disease is not endemic, only 2 were positive with the C6 ELISA. In addition, serum samples from 9/9 relapsing fever patients and 12/12 syphilitic patients also had no detectable anti-C6 antibodies. On the basis of the small number of serum samples tested thus far, the C6 peptide-based ELISA permits sensitive, specific and simple serodiagnosis of Lyme disease.. Future directions To extend our survey of crossreactivity; samples from patients with autoim mune diseases will be tested. FUNDING PUBLICATIONS None